Methods and compositions for the enzymatic production of pseudouridine triphosphate

ABSTRACT

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).

CROSS-REFERENCE TO RELATED APPLICATIONS

This International PCT application claims the benefit of and priority toU.S. Provisional Application No. 63/323,145, filed Mar. 24, 2022. Theentire specification, claims, and figures of the above-referencedapplication is hereby incorporated, in its entirety by reference.

SEQUENCE LISTING

The instant application contains contents of the electronic sequencelisting (90125-00252-Sequence-Listing; Size: 27,706 bytes; and Date ofCreation: Aug. 17, 2023) is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to the fields of chemical and enzymaticproduction of nucleotide isomers, and in particular the enzymatic andchemical production of uridine to pseudouridine (Ψ), and specificallythe and chemical and/or enzymatic production and modification ofN1-methyl-pseudouridine.

BACKGROUND

Pseudouridine-5′-triphosphate (ΨTP) is a C5-glycosidic isomer of uridinethat contains a C—C bond between the C1 of the ribose sugar and the C5of uracil, rather than the usual C1-N1 bond found in uridine.Pseudouridine is found in almost all types of non-coding RNA such astRNA, rRNA, small nuclear RNA (snRNA) as well as coding RNA, all ofwhich are generally referred to herein as “RNA,” or an “RNAoligonucleotide” meaning an oligonucleotide in which several RNAnucleosides are linked. This base modification is able to stabilize RNAand improve base-stacking by forming additional hydrogen bonds withwater through its extra amino group.

The most common modification to therapeutic RNAs is the partialsubstitution UTP with ΨTP to stabilize the RNA in the translating hostcell. Pseudo-UTP is naturally occurring, for example in tRNAs, for thisreason and is produced naturally by enzymes in diverse organisms calledPseudo-Uridine Synthases (pUS). These enzymes are responsible for themost abundant post-transcriptional modification of cellular RNA andcatalyze the site-specific isomerization of uridine residues that arealready part of an RNA oligonucleotide. Therapeutic RNAs require ΨTP asa replacement for uridine-5′-triphosphate (UTP), however the cost forsynthetically produced ΨTP is prohibitively high creating a productionbottle-neck for many RNA-based therapeutics, such as RNA-vaccines, andother RNA therapeutics and diagnostics that are increasingly produce byin vitro transcription systems. As such, there exists a need for anefficient and cost-effective method of producing ΨTP that can, inparticular, be applied to in vitro RNA production systems.

SUMMARY OF THE INVENTION

In one aspect, the invention includes systems. methods and compositionsfor the production of pseudouridine. In one embodiment, the inventioncan include systems and methods for enzymatically synthesizingpseudouridine-5′-monophosphate (ΨMP). In this embodiment, the system caninclude a substrate comprising a quantity of isolated uracil nucleobase,and a quantity of isolated ribose-5-phosphate. The substrate can beintroduced to a quantity of pseudouridine-5′-phosphate glycosidase(PsuG) enzyme, or a fragment or variant thereof, wherein said PsuGcatalyzes the formation of pseudouridine-5′-monophosphate (ΨMP) from theuracil nucleobase with a ribose-5-phosphate.

In another aspect, the invention can further include a protecting agentthat reacts with the hydroxyl groups of the ΨMP forming a protected ΨMP.The protecting agent of the invention can include an N-silyl compound,such as preferably hexamethyldisilazane (HMDS), wherein the HMDS reactswith the ΨMP forming a protected ΨMP compound as described herein.

In another aspect, the invention can further include a methylating agentthat reacts with the protected ΨMP, forming a protectedpseudouridine-5′-monophosphate (protected m1ΨMP) compound. In apreferred aspect, the methylating agent of the invention comprisesiodomethane.

In another aspect, the invention can further include a deprotectionagent that reacts with the protected m1ΨMP to remove the protectinggroup(s) thereby forming N1-methyl-pseudouridine-5′-monophosphate(m1ΨMP). In a preferred aspect, the deprotection agent of the inventionincludes a quantity of ammonia and methanol.

In another aspect, the invention can further include a phosphorylatingagent that catalyzes the sequential phosphorylation of m1ΨMP formingN1-methyl-pseudouridine-5′-diphosphate (m1ΨDP), andN1-methyl-pseudouridine-5′-triphosphate (m1ΨTP). In a preferred aspect,the phosphorylating agent of the invention includes a quantity ofPolyphosphate kinase (PPK2), or a fragment or variant thereof, and aquantity of inorganic polyphosphate, wherein said PPK2, or fragment ofvariant thereof, catalyzes the sequential phosphorylation of m1ΨMP toform m1ΨDP and m1ΨTP. In a preferred aspect, the PPK comprises a PPKfrom a thermophilic bacteria, such as for example from the genusDeinococcus or Meiothermus, or more preferably from thermophilicbacteria including Deinococcus geothermalis, Deinococcus radiodurans, orMeiothermus ruber. In still further preferred aspects, the PPK of theinvention includes a sequence according to SEQ ID NO. 4, 6, 21, or asequence having at least 80% homology with SEQ ID NO. 4, 6, or 21.

In a further aspect of the invention, the concentration of inorganicpolyphosphate is in excess, such that it promotes the forward reactionof the sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP.Moreover, in this preferred aspect, the sequential phosphorylation ofm1ΨMP by PPK is performed at a temperature, and preferably in thepresence of excess inorganic polyphosphate generating reactionconditions that causes the forward reaction of the sequentialphosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP.

In another aspect, the PsuG of the invention can include a PsuG from aan enteric bacteria, or a thermophilic bacteria, such as Deinococcusgeothermalis, and/or Escherichia coli respectively. In still furtherpreferred aspects, the PsuG of the invention can include a sequenceselected from SEQ ID NO. 1, 3, or a sequence having at least 80%homology with SEQ ID NO. 1 or 3. In another preferred embodiment, theconcentration of the uracil nucleobase and ribose-5-phosphate substratesand reaction temperature cause the reverse catalyzation of thesubstrates by the PsuG forming the ΨMP.

In another aspect, the invention includes systems, methods, andcompositions for the production of ribose-5-phosphate. In this preferredembodiment, a quantity of isolated Ribokinase (RbsK) enzyme, or afragment or variant thereof, and a quantity of ribose such that the RbsKcatalyzes the formation of said ribose-5-phosphate from said ribose inthe presence of an adenosine-triphosphate (ATP) donor, which donates aphosphate group to the ribose to form the ribose-5-phosphate and anadenosine-diphosphate (ADP). In another aspect, the RbsK if theinvention can include an RbsK from a thermophilic bacteria, such asDeinococcus geothermalis. In another aspect, the RbsK if the inventioncan include a sequence according to SEQ ID NO. 20, or a sequence havingat least 80% homology with SEQ ID NO. 20.

Additional embodiments of the invention include and isolated compoundselected from: ΨMP, protected ΨMP, protected m1ΨMP compound, m1ΨMP,m1ΨDP, and m1ΨTP produced by the methods, systems and composition of theinvention.

In another aspect the present invention includes novel systems, methods,and compositions for the enzymatic production of pseudouridine (Ψ). In apreferred aspect, Ψ, and preferably pseudouridine-5′-triphosphate (ΨTP)may be enzymatically produced from raw RNA samples, preferably extractedfrom the fermentation or cellular waste of bacterial cultures routinelygrown in laboratories or yeast waste product from the beer and winefermentation industry. In this aspect, RNA is isolated from thefermentation waste and enzymatically converted into a Ψ form, which isfurther enzymatically converted into its mono-phosphate form (ΨMP) priorto tri-phosphate regeneration. In one preferred aspect, tri-phosphateregeneration is accomplished using inorganic polyphosphates (PPi) andadenosine mono-phosphate (AMP) as part of a dual enzyme system, whichmay preferably include adenosine kinase (AdK) and polyphosphate kinase(PPK) to regenerate ΨTP from ΨMP.

In a preferred aspect of the invention, N1-methyl-pseudouridinetri-phosphate (m1Ψ) may be generated from isolated uridine continuingRNA oligonucleotides. In this preferred aspect, RNA containing uridinemay be isolated from, for example, fermentation waste. The uridineresidues of the RNA oligonucleotide may be converted topseudouridine-5′-triphosphate (Ψ) by pseudouridine synthase (pUS). Next,the Ψ residues may be methylated forming m1Ψ residues byN1-pseudouridine methyltransferase which can be further digested intonucleotide mono-phosphates (m1ΨMP), preferably by a nuclease, such as P1or a 5′-Phosphodiesterase (5′-PDase). The resulting m1ΨMP can bepurified and enzymatically converted into m1Ψ tri-phosphate by anucleoside diphosphate kinase (NdK), such as adenosyl kinase in thepresence of an adenosine-triphosphate (ATP) donor.

In another preferred embodiment, the invention includes a modifiedN1-pseudouridine methyltransferase, or a fragment or variant thereof,that may further catalyze the methylation of the newly formed Ψ residuesin a RNA oligonucleotide forming a series of N1-methyl-pseudouridine(m1Ψ) residues. In one preferred embodiment, the 129R and 132R residesof an exemplary N1-pseudouridine methyltransferase Nep1 are converted to129A and 132A forming a modified N1-pseudouridine methyltransferase(mNep1). These engineered mutations may allow the formation ofindividual N1-methyl-pseudouridine (m1Ψ) residues from the Ψ residues ofthe RNA oligonucleotide by reducing the coordination of mNep1 with thenucleotide adjacent to the pUTP residues in the RNA oligonucleotides.

In another aspect, the present invention includes alternative novelsystems, methods, and compositions for the enzymatic production of ΨTPutilizing a pseudouridine-5′-phosphate glycosidase (PsuG) enzyme. Inthis preferred aspect, PsuG may be used to catalyze the formation ofpseudouridine-5′-monophosphate (ΨMP) from a uracil nucleobase with aribose-5-phosphate substrate. The temperature and substrateconcentrations of this reaction cause the reverse action of the PsuGenzyme which, under normal conditions, catalytically cleaves a ΨMPsubstrate to form uracil and ribose-5-phosphate. The resulting ΨMP canbe enzymatically converted into Ψ tri-phosphate by a nucleosidediphosphate kinase (NdK), such as adenosyl kinase (Adk) in the presenceof an adenosine-triphosphate (ATP) donor. Alternatively, the resultingΨMP can be enzymatically converted into Ψ tri-phosphate (ΨTP) by adeoxynucleoside kinase, such as PPK2, in the presence of a phosphatedonor, which may preferably include sodium hexamethaphosphate. The ΨTPmay further be optionally methylated forming m1ΨTP.

Additional aspects of the invention may be evidenced from thespecification, claims and figures provided below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . shows a schematic scheme for the enzymatic production of ΨTP,and in particular m1-pseudouridine-5′-triphosphate (m1ΨTP) in oneembodiment thereof.

FIG. 2 . shows expression and properties of recombinantly expressedpseudouridine glycosidase (PsuG) enzyme in one embodiment thereof.

FIG. 3A-B. shows (A) a schematic scheme for the enzymatic production ofuridine-5′-monophosphate (ΨMP) from uracil and ribose-5-phosphatecatalyzed by the reverse reaction of PsuG in one embodiment thereof; (B)shown time-course production of ΨMP catalyzed by PsuG in one embodimentthereof.

FIG. 4A-B. shows (A) a schematic scheme for the enzymatic regenerationof ΨTP from ΨMP using sodium hexametaphosphate catalyzed bydeoxynucleoside kinase (PPK2) in one embodiment thereof; (B) theenzymatic regeneration of ΨTP from ΨMP using an enzymatic feedback loopof PPK2+inorganic polyphosphate and a nucleoside-diphosphate kinases(NdK) to regenerate ΨTP from ΨMP using an ATP donor, resulting in theproduction of AMP which is recycled back to ATP in one embodimentthereof.

FIG. 5 . show stepwise scheme 1 for the enzymatic and chemicalproduction and m1ΨTP in one embodiment thereof.

FIG. 6 . shows enzymatic production of ΨMP using the scheme of FIG. 5 ,where ribose and polyP were dissolved in ˜500 ml H2O and adjusted to pH7 with the solution described in the Figure being fed into the reactionover ˜20 hr.

FIG. 7 . demonstrates stepwise chemical methylation of ΨMP forming1mΨMP. As shown in the Figures, small aliquot of chemical methylationreaction (200 mL) taken out after 36h, solvent removed under stream ofnitrogen, deprotected using 300 mL of 7M NH₃ in MeOH, solvent removedagain, dissolved in 500 mL of aq. 10 mM DBAA, 5 mL of that solution,subject to LC-MS analysis.

FIG. 8 . shows enzymatic production of 1mΨTP from 1mΨMP. Reactionconditions included 25 mM 1mΨTP, 10 mM polyp, 0.25 mM PPK2, buffer: 50mM HEPES, 25 mM Mg²⁺, and 1 mM Mn²⁺, at pH 7.

FIG. 9A-B. shows HPLC and MS analyses of purified 1mΨTP synthesized byscheme 1 as described herein.

FIG. 10 . shows in vitro transcription of RNA using 1mΨTP synthesized byscheme 1 as described herein as compared to commercially availabletranscription products.

FIG. 11A-B. shows (A) HPLC analysis of m1Ψ run against standard (B)demonstrates HPLC analysis of the amounts and conversion of 1mΨTP, 1mΨDPand 1mΨMP from in vitro RNA transcription mixture.

FIG. 12A-B. shows experimental results demonstrating enzymatic (A)capping and (B) tailing of mRNA transcripts utilizingN1-Methylpseudouridine generated by the method(s) of the invention inone embodiment thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes novel systems, methods, and compositionsfor the scalable enzymatic production of pUTP from uridine continuingRNA oligonucleotides, and preferably RNA oligonucleotides isolated fromfermentation waste and other natural or synthetic sources. In apreferred embodiment, the invention may include generating a samplecontaining an RNA oligonucleotide, and preferably a plurality of RNAoligonucleotides isolated from fermentation waste, such as bacterialcultures grown in laboratories or macro-molecule production systems, oryeast waste products from the beer and wine fermentation industry, aswell as yeast-based macro-molecule production systems and the like.

Preferably in an in vitro reaction chamber, such as a bio-chamber, anisolated pseudouridine synthase enzyme, or a fragment or variantthereof, may catalytically convert uridine nucleotide residues in theRNA oligonucleotides into Ψ residues. In a preferred embodiment, apseudouridine synthase of the invention may selected from the groupconsisting of: SEQ ID NO's. PUS1-PUS9 (SEQ ID NO.'s 11-19), or afragment or variant thereof. In the same or separate in vitro reactionchamber, an N1-pseudouridine methyltransferase, or a fragment or variantthereof, may further catalyze the methylation of the newly formed Ψresidues in the RNA oligonucleotides forming a series ofN1-methyl-pseudouridine (m1Ψ) residues. In a preferred embodiment,N1-pseudouridine methyltransferase comprises Nep1 and more preferablymay include a mNep1 enzyme according to SEQ ID NO. 8, or a fragment orvariant thereof.

In another preferred embodiment, in the same or separate in vitroreaction chamber, an modified N1-pseudouridine methyltransferase(mNep1), or a fragment or variant thereof, may catalyze the methylationof the newly formed Ψ residues in the RNA oligonucleotides forming aseries of N1-methyl-pseudouridine (m1Ψ) residues. In this embodiment,the 129R and 132R resides of Nep1 are converted to 129A and 132A formingmNep1 (SEQ ID NO. 9). These engineered mutations may allow the formationof individual m1Ψ residues from the Ψ residues of the RNAoligonucleotide by reducing the coordination of mNep1 with thenucleotide(s) adjacent to the Ψ residues in the RNA oligonucleotides.

The RNA oligonucleotide now containing a series of m1Ψ residues may bedigested forming a plurality of nucleotide mono-phosphates, includingm1Ψ-monophosphates (m1ΨMP). In a preferred embodiment, the RNAoligonucleotide may be digested with a nuclease P1, or5′-Phosphodiesterase (5′-PDase), or a fragment or variant thereof. Theresulting m1ΨMP can further be isolated and/or purified, for examplethrough a weak anion exchange column, or other similar purificationapparatus.

The enzymatically generated m1ΨMP may further be regenerated into theirN1-methyl-pseudouridine-5′-triphosphate (m1ΨTP) form. In this preferredembodiment, a nucleoside diphosphate kinase (NdK), in the presence of anadenosine-triphosphate (ATP) donor, catalyzes the transfer of aphosphate group to the m1ΨMP, forming adenosine-monophosphate (AMP)while sequentially forming a m1Ψ-di-phosphate (m1ΨDP), and ultimately apseudouridine-5′-triphosphate (m1ΨTP) that can be further isolatedand/or purified. In one embodiment, the NdK of the invention may includean adenosyl kinase (AdK), and may preferably include an AdK accordingto: SEQ ID NO. 10, or a fragment or variant thereof.

The AMP resulting from the ATP donor of the invention may further beregenerated through the action of a polyphosphate kinase (PPK). In thisembodiment, an AMP may be regenerated back into an ATP by the transferof a inorganic polyphosphate to the AMP by a PPK enzyme. In a preferredembodiment, the PPK enzyme of the invention may include a PPK2 enzymeselected from the group consisting of: SEQ ID NO's. 4, 6, or a fragmentor variant thereof. The regenerated ATP may be recycled back into thereaction cycle regenerating m1ΨTP from m1ΨMP.

As noted above, the above reactions may occur in vitro, separately or inthe same or sequential in vitro reaction chambers. In a preferredembodiment, the above reactions may be performed in an in vitrotranscription system, such as a cell-free expression system, or an invitro RNA production system.

The present invention includes novel systems, methods, and compositionsfor the enzymatic production of Ψ derived from a uracil nucleobase and aribose-5-phosphate. In a preferred embodiment,pseudouridine-5′-phosphate glycosidase (PsuG) enzyme may catalyze theformation of pseudouridine-5′-monophosphate (ΨMP) from the reversiblereaction of two substrates, namely uracil nucleobase with aribose-5-phosphate, preferably in an in vitro system, such as an invitro transcription, or RNA production system. Substrate and temperaturecondition may be modulated to cause the reverse reaction of PsuG therebycatalyzing the formation ΨMP from the uracil nucleobase andribose-5-phosphate substrates, where the normally forward reactionproceeds in the opposite direction catalyzing the cleavage of a ΨMPsubstrate to form a uracil nucleobase a ribose-5-phosphate product. Inthis embodiment, the revere reaction of PsuG may be facilitated byelevating the temperature of the reaction, for approximately 37° C. to50° C., and increasing concentrations of both substrates, namely uraciland ribose-5-phosphate. In one embodiment, the concentrations of bothuracil and ribose-5-phosphate may be at least 1 millimolar each. In thispreferred embodiment, PsuG may be selected from a thermophilic bacteria,such as D. geothermalis (SEQ ID NO. 1, or 2) allowing the reaction to berun at a higher temperature, and preferably at least 50° C.

As noted above, the invention may include the generation of ΨTP from ΨMPby contacting the ΨMP with PPK2 in the present of an inorganicpolyphosphate source, such as sodium hexamethaphosphate. In analternative embodiment, the enzymatically produced ΨMP may beregenerated to form ΨTP through the catalytic action of a nucleosidediphosphate kinase (NdK) in the presence of an adenosine-triphosphate(ATP) donor, with the resulting ATP donor, now an AMP, being regeneratedas described above.

The enzymatically created ΨTP may further be methylated by aN1-pseudouridine methyltransferase, or a fragment or variant thereof,forming N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP). In a preferredembodiment, N1-pseudouridine methyltransferase includes Nep1 (SEQ ID NO.8).

In one embodiment the invention includes systems, methods, andcompositions for the enzymatic and chemical production of m1ΨTP, andpreferably its use in an in vitro transcription reaction to generate RNAincorporating the same. Generally referring to the scheme provided inFIG. 5 , invention can include the enzymatic synthesis of Ψ derived froma uracil nucleobase and a ribose-5-phosphate. In one embodiment, aRibokinase (RbsK) enzyme can convert ribose to ribose-5-phosphate, withthe phosphate group being donated from a molecule of ATP which isconverted into ADP. As shown in FIG. 5 , the ADP can be regenerated byreacting with a PPK enzyme in the presence of a polyphosphate formingATP which can go on to be coupled to a ribose by RbsK formingribose-5-phosphate. In a preferred embodiment, a Ribokinase (RbsK)enzyme according to the amino acid sequence SEQ ID NO. 20, or a fragmentof variant thereof, can convert ribose to ribose-5-phosphate, with thephosphate group being donated from a molecule of ATP which is convertedinto ADP. Again, the resulting ADP can be regenerated by reacting with aPPK enzyme according to SEQ ID NO. 4, 6, 21, or a fragment of variantthereof, in the presence of a polyphosphate forming ATP which can go onto be coupled to a ribose by RbsK according to the amino acid sequenceSEQ ID NO. 20, or a fragment of variant thereof, formingribose-5-phosphate.

Again referring to synthesis scheme of FIG. 5 , the present inventionfurther includes novel systems, methods, and compositions for theenzymatic production of Ψ derived from a uracil nucleobase and aribose-5-phosphate, preferably generated by the methods described above.In a preferred embodiment, a pseudouridine-5′-phosphate glycosidase(PsuG) enzyme may catalyze the formation ofpseudouridine-5′-monophosphate (ΨMP) from the reversible reaction of twosubstrates, namely uracil nucleobase with a ribose-5-phosphate,preferably in an in vitro system, such as an in vitro transcription, orRNA production system. Substrate and temperature condition may bemodulated to cause the reverse reaction of PsuG thereby catalyzing theformation ΨMP from the uracil nucleobase and ribose-5-phosphatesubstrates, where the normally forward reaction proceeds in the oppositedirection catalyzing the cleavage of a ΨMP substrate to form a uracilnucleobase a ribose-5-phosphate product. In this embodiment, the reversereaction of PsuG may be facilitated by elevating the temperature of thereaction, for approximately 37° C. to 50° C., and increasingconcentrations of both substrates, namely uracil and ribose-5-phosphate.In one embodiment, the concentrations of both uracil andribose-5-phosphate may be at least 1 millimolar each. In this preferredembodiment, PsuG may be selected from a thermophilic bacteria, such asD. geothermalis, and may include the amino acid sequence according toSEQ ID NO. 1, or 2, or a fragment of variant thereof. In this manner,the use of enzymes from thermophilic organisms allows the reaction to berun at a higher temperature, and preferably at least 50° C.

Again referring to the synthesis scheme of FIG. 5 , the hydroxyl groupsof the ΨMP substrate formed by a uracil nucleobase a ribose-5-phosphateis methylated to form m1ΨMP. In one embodiment, the hydroxyl groups ofthe ΨMP can react with a protecting agent, preferably an N-silylcompound, such as a quantity of hexamethyldisilazane (HMDS) generating aprotecting group of trimethylsilyl ether (—OTMS) at the former hydroxylposition of the ΨMP, forming what is referred to herein as a protectedΨMP, according to the formula provided below:

The resulting protected ΨMP compound can react with a methylating agentso as to be methylated at the 1 position, forming a protected m1ΨMPcompound. In a preferred embodiment, the protected m1ΨM is reacted witha quantity of a methylating agent, and preferably iodomethane thatmethylates the 1 position of the protected ΨMP, forming a protected m1ΨMaccording to the formula provided below:

The protected m1ΨMP compound of the invention can further bedeprotected. In this embodiment, a deprotection agent can react with theprotected m1ΨMP forming a m1ΨMP. In one embodiment, for example m1ΨMPwith a deprotection agent that includes a quantity of ammonia inmethanol. Removal of the protecting groups results in the formation ofm1ΨMP.

In a preferred embodiment, the m1ΨMP synthesized by the proceeding stepscan be enzymatically converted to m1ΨTP by diphosphorylation mediated bya phosphorylating agent, which in a preferred embodiment can includes aPPK enzyme, and preferably a PPK2 enzyme derived from thermophilicbacteria. Again referring to FIG. 5 , a phosphate group from ATP istransferred to m1ΨMP, forming m1ΨDP, followed by the transfer of anotherphosphate group from an ATP to m1ΨDP, forming m1ΨTP respectively. Inthis preferred embodiment, the diphosphorylation is mediated by a PPKenzyme, and preferably a PPK2 enzyme to according to SEQ ID NO. 4, 6,21, or a fragment of variant thereof.

In certain embodiment, the invention further include systems, methodsand compositions for the production of heterologous proteins for use inthe production of m1ΨTP. For example, in one embodiment, a cell, andpreferably a yeast, bacterial or other prokaryotic cell can betransformed by an expression vector expressing one or more of theenzymes of the invention. In this embodiment, a cell can express aheterologous nucleotide, operably linked to a promoter, encoding a PsuG,and/or PPK2 protein, or a fragment of variant thereof of the invention.In another preferred embodiment, a cell, such as a yeast cell, canexpress a heterologous nucleotide, operably linked to a promoter,encoding a PsuG enzyme according to SEQ ID NO. 1, or 2, or a fragment ofvariant thereof, and/or PPK2 enzyme to according to SEQ ID NO. 4, 6, 21,or a fragment of variant thereof. In this preferred embodiment, thetransformed yeast cell can be cultured and in a suitable media.Expression of the PsuG and/or PPK2 enzyme can occur, with the resultingproteins being actively or passively directed out of the yeast cell intothe supernatant, from which they can be isolated.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and CurrentProtocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons,Inc. 2001. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

Unless otherwise required by context, the use herein of the singular isto be read to include the plural and vice versa. The term “a” or “an”used in relation to an entity is to be read to refer to one or more ofthat entity. As such, the terms “a” (or “an”), “one or more,” and “atleast one” are used interchangeably herein.

As used herein, the term “comprise,” or variations thereof such as“comprises” or “comprising,” are to be read to indicate the inclusion ofany recited integer (e.g., a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g., features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein the term “comprising” is inclusive oropen-ended and does not exclude additional, unrecited integers ormethod/process steps.

The term “polynucleotide” or “nucleotide” as used herein indicates anorganic polymer composed of two or more monomers including nucleotides,nucleosides or analogs thereof. The term “nucleotide” refers to any ofseveral compounds that consist of a ribose or deoxyribose sugar joinedto a purine or pyrimidine base and to a phosphate group and that is thebasic structural unit of nucleic acids. The term “nucleoside” refers toa compound (such as guanosine or adenosine) that consists of a purine orpyrimidine base combined with deoxyribose or ribose and is foundespecially in nucleic acids. The term “nucleotide analog” or “nucleosideanalog” refers respectively to a nucleotide or nucleoside in which oneor more individual atoms have been replaced with a different atom or awith a different functional group. Accordingly, the term“polynucleotide” includes nucleic acids of any length, and in particularDNA, RNA, analogs and fragments thereof. A polynucleotide of three ormore nucleotides is also called an “oligomer” or “oligonucleotide.”

The term “messenger ribonucleic acid” (messenger RNA, mRNA) refers to aribonucleic acid (RNA) molecule that mediates the transfer of geneticinformation to ribosomes in the cytoplasm, where it serves as a templatefor protein synthesis. It is synthesized from a DNA template during theprocess of transcription. A “ribonucleic acid” (RNA) is a polymer ofnucleotides linked by a phosphodiester bond, where each nucleotidecontains ribose or a modification thereof as the sugar component. Eachnucleotide contains an adenine (A), a guanine (G), a cytosine (C), anuracil (U) or a modification thereof as the base. The geneticinformation in a mRNA molecule is encoded in the sequence of thenucleotide bases of the mRNA molecule, which are arranged into codonsconsisting of three nucleotide bases each. Each codon encodes for aspecific amino acid of the polypeptide, except for the stop codons,which terminate translation (protein synthesis). Within a living cell,mRNA is transported to a ribosome, the site of protein synthesis, whereit provides the genetic information for protein synthesis (translation).For a fuller description, see, Alberts B et al. (2007) Molecular Biologyof the Cell, Fifth Edition, Garland Science.

As used herein, “in vitro transcription” (IVT) or “RNA productionsystem” refers to a cell-free reaction in which a double-stranded DNA(dsDNA) template is copied by a DNA-directed RNA polymerase (typically abacteriophage polymerase) to produce a product that contains RNAmolecules copied from the template. An example of in vitro transcriptionmay include cell-free expression systems that produce RNA transcripts orother macromolecules, such as peptides. In certain embodiments, theinvention may encompass the in vitro production of artificial mRNA aswell as wild-type mRNA. An artificial mRNA (sequence) may typically beunderstood to be an mRNA molecule, that does not occur naturally. Inother words, an artificial mRNA molecule may be understood as anon-natural mRNA molecule. Such mRNA molecule may be non-natural due toits individual sequence (which does not occur naturally) and/or due toother modifications, e.g., structural modifications of nucleotides whichdo not occur naturally. Typically, artificial mRNA molecules may bedesigned and/or generated by genetic engineering methods to correspondto a desired artificial sequence of nucleotides (heterologous sequence).In this context an artificial sequence is usually a sequence that maynot occur naturally, i.e., it differs from the wildtype sequence by atleast one nucleotide. The term “wild type” may be understood as asequence occurring in nature. Further, the term “artificial nucleic acidmolecule” is not restricted to mean “one single molecule” but is,typically, understood to comprise an ensemble of identical molecules.Accordingly, it may relate to a plurality of identical moleculescontained in an aliquot.

In certain embodiment, the invention may encompass the in vitroproduction of bi/multicistronic mRNA: mRNA, that typically may have two(bicistronic) or more (multi cistronic) open reading frames (ORF)(coding regions or coding sequences). An open reading frame in thiscontext is a sequence of several nucleotide triplets (codons) that canbe translated into a peptide or protein. Translation of such an mRNAyields two (bicistronic) or more (multi cistronic) distinct translationproducts (provided the ORFs are not identical). For expression ineukaryotes such mRNAs may for example comprise an internal ribosomalentry site (IRES) sequence.

In one embodiment, the in vitro produce mRNA configured to be translatedto form a peptide, and preferably in a host organism, such as a mammalor human subject in need thereof. A peptide is a polymer of amino acidmonomers. Usually, the monomers are linked by peptide bonds. The term“peptide” does not limit the length of the polymer chain of amino acids.In some embodiments of the present invention a peptide may for examplecontain less than 50 monomer units. Longer peptides are also calledpolypeptides, typically having 50 to 600 monomeric units, morespecifically 50 to 300 monomeric units.

Additional examples of IVT systems, include in vitro recombinantcell-free expression systems, which refers to the cell-free synthesis ofpolypeptides in a reaction mixture or solution comprising biologicalextracts and/or defined cell-free reaction components, such as theexemplary system described by Koglin et al., in PCT/US2020/028005 andPCT/US2021/027774 (incorporated herein by reference). The reactionmixture may optionally comprise a template, or genetic template, forproduction of the macromolecule, e.g., DNA, mRNA, etc.; monomers for themacromolecule to be synthesized, e.g., amino acids, nucleotides, etc.;and such co-factors, enzymes and other reagents that are necessary forthe synthesis, e.g., ribosomes, tRNA, polymerases, transcriptionalfactors, etc. The recombinant cell-free synthesis reaction, and/orcellular adenosine triphosphate (ATP) energy regeneration systemcomponents, incorporated by reference herein, may be performed/added asbatch, continuous flow, or semi-continuous flow.

Examples of in vitro production systems have been also previouslydescribed in the art, including U.S. Pat. No. 11,136,586, and PCTApplication No. PCT/US2020/028005. The disclosed methods and conditionsfor the production of synthetic RNAs in each of the aforementionedreferences, including the methods, systems and compositions outline inthe claims, Examples and Materials and Methods is hereby incorporated intheir entirety by reference. For example, a synthetic RNAoligonucleotide may be generated and capped within a Cell Free (CF)expression system. In one embodiment, a CF expression system may includea fully recombinant stable, reliable, and functional in vitrotranscription system for the continuous flow production of RNA. As notedabove, an exemplary CF system being generally described by A. Koglin andM. Humbert et al., in PCT/US2018/0121121, and PCT/US2021/027774(previously identified as incorporated by reference) may be used as anin vitro platform to produce synthetic mRNAs. As noted in the art,lysate-based in vitro systems are challenged by limited stability oftypical E. coli enzymes, by the activity of most metabolic processes(nucleotide recycling) and the presence of nucleases and proteases andinsufficient ATP regeneration. Utilizing the CF system described byKoglin and Humbert, and using only components: linear DNA template, anaffinity-tagged RNA polymerase, nucleotides in a defined buffer system,and a capping enzyme of the invention, the in vitro synthesis of themRNA may be performed in hollow fiber reactors using a continuous flowsystem, as well as other traditional bioreactors known in the art. Usingthis in vitro system, the inner chamber (hollow fibers) of thebioreactor provides additional nucleotides in flow, the outer chamberholds the RNA polymerase and each linear DNA template. In this setup,the present inventors demonstrate that the total turnover of the RNAP isat least 50 fold higher than in batch reaction and, coupled withmodifications to selected enzyme, produce cleaner mRNA without smear.All enzymes are engineered with an affinity tag, to allow the wholereaction to be washed through a bed of affinity resin, which ispartially loaded with DNase to remove the template and to capture theRNA polymerase. In this way the process avoids the need to addressphenol precipitations and spin column purifications (which is still anissue with traditional vaccine processes). In this system, the leachingor carryover of components from the RNA biosynthesis may be the mid ppbrange. After a scalable and simple precipitation and drying process, theresulting mRNA is stable as a powder and does not contain traces of anycomponents from the manufacturing process. It is ready to ship requiringonly reduced volumes without the needed of hard-to-monitor and expensiveshipping conditions.

In some embodiments the IVT of the invention may include a “bioreactor”that may be any form of enclosed apparatus configured to maintain anenvironment conducive to the production of macromolecules in vitro. Abioreactor may be configured to run on a batch, continuous, orsemi-continuous basis, for example by a feeder reaction solution.Examples of a bioreactor and conditions for synthesis of RNA or othermacromolecules has been previously described in by Koglin, et al.PCT/US2021/027774.

The terms “isolated”, “purified”, or “biologically pure” as used herein,refer to material that is substantially or essentially free fromcomponents that normally accompany the material in its native state orwhen the material is produced. In an exemplary embodiment, purity andhomogeneity are determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high-performance liquidchromatography. A nucleic acid or particular bacteria that are thepredominant species present in a preparation is substantially purified.In an exemplary embodiment, the term “purified” denotes that a nucleicacid or protein that gives rise to essentially one band in anelectrophoretic gel. Typically, isolated nucleic acids or proteins havea level of purity expressed as a range. The lower end of the range ofpurity for the component is about 60%, about 70% or about 80% and theupper end of the range of purity is about 70%, about 80%, about 90% ormore than about 90%.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form,” as used herein, pertains to a compound in which one ormore reactive functional groups are protected from undesirable chemicalreactions, that is, are in the form of a protected or protecting group(also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. As used herein “protecting group” means anatomic group that, when attached to a reactive functional group in amolecule, masks, reduces or prevents the reactivity of the functionalgroup. Non-limiting examples of protecting groups may be found in“Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts,ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York. A “deprotectingagent,” is any compound, or mixture of compounds that removes aprotecting group.

In a preferred embodiment, a protecting group can include hydroxy“protecting group,” which can be any protecting group suitable for ahydroxy functional group. Representative hydroxy protecting groupsinclude, but are not limited to, silanes, ethers, esters, or others.Representative hydroxy protecting groups include, but are not limited tohexamethyldisilazane (HMDS), trimethyl silane (TMS), t-butyl dimethylsilane (TBDMS), t-butyl diphenyl silane (TBDPS), methoxy-methyl (MOM),tetrahydropyran (THP), t-butyl, allyl, benzyl, acetyl, pivaloyl, orbenzoyl. In some embodiments, the hydroxy protecting group can betrimethyl silane (TMS), t-butyl dimethyl silane (TBDMS), t-butyldiphenyl silane (TBDPS), methyl-methoxy (MOM), tetrahydropyran (THP),t-butyl, allyl, benzyl, acetyl, pivaloyl, or benzoyl. In someembodiments, the hydroxy protecting group can be benzyl. In someembodiments, the hydroxy protecting group can be TBS.

“Methylating agent” means a reactive species, having electrophilicproperties, which is capable of introducing a “methyl group” at thenitrogen atom of naltrexone, so as to form a covalent bond therewith.Illustrative methylating agents can be represented by the formula CH3Z,wherein “Z” is a leaving group which, upon its departure, enables CH3 toform a covalent bond with the nitrogen atom of naltrexone, forming MNTX.Methylating agents in general, and leaving groups in general, are wellknown to those of ordinary skill in the art and are describedextensively in both the patent literature and in chemistry text books.Suitable Z groups include, but are not limited to, fluoro, chloro,bromo, iodo, iodomethane, —OSO2CF3, CH3OSO2O—, —OSO2CH3,—OSO2C6H4-p-CH3, —OSO2C6H4-p-Br.

As the phosphorylating agent, means reagents or enzymes that aregenerally used in the phosphorylation of a hydroxyl group. Examples ofsuch phosphorylating agent compounds include diesters of phosphoric acidsuch as dibenzyl phosphate and the like; dithioesters of phosphoric acidsuch as monocyclohexylammonium S,S′-diphenylphosphoro dithioate and thelike; phosphoric acid chlorides such as phosphoryl chloride, diallylchlorophosphonate and the like. As additives, for example, azo compoundssuch as diethyl azodicarboxylate, diisopropyl azodicarboxylate and thelike; phosphines such as triphenylphosphine and the like; allenesulfonicacid chlorides such as 2,4,6-triisopropylbenzenesulfonic acid chlorideand the like, bases such as pyridine, tert-butylmagnesium chloride andthe like can be referred to. Examples of such phosphorylating agents canalso refer to enzymes, such as PPK2.

In preferred embodiments, the output of the cell-free expression systemmay be a product, RNA, or other macromolecule such as a peptide orfragment thereof that may be isolated or purified. In this embodiment,solation or purification of a of a target protein wherein the targetprotein is at least partially separated from at least one othercomponent in the reaction mixture, for example, by organic solventprecipitation, such as methanol, ethanol or acetone precipitation,organic or inorganic salt precipitation such as trichloroacetic acid(TCA) or ammonium sulfate precipitation, nonionic polymer precipitationsuch as polyethylene glycol (PEG) precipitation, pH precipitation,temperature precipitation, immunoprecipitation, chromatographicseparation such as adsorption, ion-exchange, affinity and gel exclusionchromatography, chromatofocusing, isoelectric focusing, high performanceliquid chromatography (HPLC), gel electrophoresis, dialysis,microfiltration, and the like.

The term “nucleic acid” as used herein refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, andpeptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”,“polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleicacid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acidfragment”, and “isolated nucleic acid fragment” are used interchangeablyherein. For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). Estimates are typically derived from agarose oracrylamide gel electrophoresis, from sequenced nucleic acids, or frompublished DNA sequences. For proteins, sizes are given in kilodaltons(kDa) or amino acid residue numbers. Proteins sizes are estimated fromgel electrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

As is known in the art, different organisms preferentially utilizedifferent codons for generating polypeptides. Such “codon usage”preferences may be used in the design of nucleic acid molecules encodingthe proteins and chimeras of the invention in order to optimizeexpression in a particular host cell system. All nucleotide sequencesdescribed in the invention may be codon optimized for expression in aparticular organism, or for increases in production yield. Codonoptimization generally improves the protein expression by increasing thetranslational efficiency of a gene of interest. The functionality of agene may also be increased by optimizing codon usage within the customdesigned gene. In codon optimization embodiments, a codon of lowfrequency in a species may be replaced by a codon with high frequency,for example, a codon UUA of low frequency may be replaced by a codon CUGof high frequency for leucine. Codon optimization may increase mRNAstability and therefore modify the rate of protein translation orprotein folding. Further, codon optimization may customizetranscriptional and translational control, modify ribosome bindingsites, or stabilize mRNA degradation sites.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), the complementary (or complement)sequence, and the reverse complement sequence, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); andRossolini et al., Mol. Cell. Probes 8:91-98 (1994)). In addition to thedegenerate nature of the nucleotide codons which encode amino acids,alterations in a polynucleotide that result in the production of achemically equivalent amino acid at a given site, but do not affect thefunctional properties of the encoded polypeptide, are well known in theart. “Conservative amino acid substitutions” are those substitutionsthat are predicted to interfere least with the properties of thereference polypeptide. In other words, conservative amino acidsubstitutions substantially conserve the structure and the function ofthe reference protein. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,can also be expected to produce a functionally equivalent protein orpolypeptide. Exemplary conservative amino acid substitutions are knownby those of ordinary skill in the art. Conservative amino acidsubstitutions generally maintain (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a beta sheetor alpha helical conformation, (b) the charge or hydrophobicity of themolecule at the site of the substitution, and/or (c) the bulk of theside chain. Further disclosure of a nucleotide sequence, specificallyincludes the resulting amino acid sequence for which it encodes and viceversa.

As used herein, the term “transformation” or “genetically modified”refers to the transfer of one or more nucleic acid molecule(s) into acell, preferably through an expression vector. A microorganism is“transformed” or “genetically modified” by a nucleic acid moleculetransduced into the bacteria or cell or organism when the nucleic acidmolecule becomes stably replicated. As used herein, the term“transformation” or “genetically modified” encompasses all techniques bywhich a nucleic acid molecule can be introduced into a cell or organism,such as a bacteria.

As used herein, the term “promoter” refers to a region of DNA that maybe upstream from the start of transcription, and that may be involved inrecognition and binding of RNA polymerase and other proteins to initiatetranscription. A promoter may be operably linked to a coding sequencefor expression in a cell, or a promoter may be operably linked to anucleotide sequence encoding a signal sequence which may be operablylinked to a coding sequence for expression in a cell.

The term “operably linked,” when used in reference to a regulatorysequence and a coding sequence, means that the regulatory sequenceaffects the expression of the linked coding sequence. “Regulatorysequences,” or “control elements,” refer to nucleotide sequences thatinfluence the timing and level/amount of transcription, RNA processingor stability, or translation of the associated coding sequence.Regulatory sequences may include promoters; translation leadersequences; introns; enhancers; stem-loop structures; repressor orbinding sequences; termination sequences; polyadenylation recognitionsequences; etc. Particular regulatory sequences may be located upstreamand/or downstream of a coding sequence operably linked thereto. Also,particular regulatory sequences operably linked to a coding sequence maybe located on the associated complementary strand of a double-strandednucleic acid molecule.

The term “expression,” as used herein, or “expression of a codingsequence” (for example, a gene or a transgene) refers to the process bywhich the coded information of a nucleic acid transcriptional unit(including, e.g., genomic DNA or cDNA) is converted into an operational,non-operational, or structural part of a cell, often including thesynthesis of a protein. Gene expression can be influenced by externalsignals; for example, exposure of a cell, tissue, or organism to anagent that increases or decreases gene expression. Expression of a genecan also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

An “expression vector” is nucleic acid capable of replicating in aselected host cell or organism, or in vitro environment, such as acell-free expression system or other IVT system. An expression vectorcan replicate as an autonomous structure, or alternatively canintegrate, in whole or in part, into the host cell chromosomes or thenucleic acids of an organelle, or it is used as a shuttle for deliveringforeign DNA to cells, and thus replicate along with the host cellgenome. Thus, an expression vector are polynucleotides capable ofreplicating in a selected host cell, organelle, or organism, e.g., aplasmid, virus, artificial chromosome, nucleic acid fragment, and forwhich certain genes on the expression vector (including genes ofinterest) are transcribed and translated into a polypeptide or proteinwithin the cell, organelle or organism; or any suitable construct knownin the art, which comprises an “expression cassette.” In contrast, asdescribed in the examples herein, a “cassette” is a polynucleotidecontaining a section of an expression vector of this invention. The useof the cassettes assists in the assembly of the expression vectors. Anexpression vector is a replicon, such as plasmid, phage, virus, chimericvirus, or cosmid, and which contains the desired polynucleotide sequenceoperably linked to the expression control sequence(s).

The terms “expression product” as it relates to a protein expressed in acell-free expression system as generally described herein, are usedinterchangeably and refer generally to any peptide or protein havingmore than about 5 amino acids. The polypeptides may be homologous to, ormay be exogenous, meaning that they are heterologous, i.e., foreign, tothe organism from which the cell-free extract is derived, such as ahuman protein, plant protein, viral protein, yeast protein, etc.,produced in the cell-free extract.

In some embodiments, the term a nucleic acid or peptide may be from asource, such as a virus. In this context a “derived” nucleic acid, suchas RNA, or peptide means extracted from, or expressed and isolated froma bacteria, eukaryotic cell or other source, such as fermentation waste.For example, in one embodiment a capping protein may be derived from anexpression vector expressed in a bacteria, or eukaryotic cell.

As used herein, “RNA sample” or “sample” refers to a composition thatcontain one, or a plurality of oligonucleotides containing uridineresidues. An RNA sample may comprise a naturally occurring RNA (e.g.,extracted from a cell, tissue, or organism), a RNA produced by in vitrotranscription, and/or a chemically synthesized RNA, or an RNA sampleharvest from fermentation waste culture, and/or cells.

As used herein, “variant” refers to a protein that has an amino acidsequence that is different from a naturally occurring amino acidsequence (i.e., having less than 100% sequence identity to the aminoacid sequence of a naturally occurring protein) but that is at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98% or at least 99% identical to the naturally occurring amino acidsequence.

As used herein, “fragment” refers to a portion of a peptide ornucleotide sequence that still retains the activity of the whole.

Unless other stated, disclosure of a DNA sequence also include thecorresponding RNA and amino acid sequence including all redundant codonsand conservative amino acid substitutions, disclosure of a RNA sequencealso include the corresponding DNA and amino acid sequence including allredundant codons and conservative amino acid substitutions, and finallydisclosure of amino acid sequence also include the corresponding RNA andDNA sequence including all redundant codons and conservative amino acidsubstitutions and vice versa.

Additional Embodiments

In one preferred embodiment, the invention includes a method producingpseudouridine comprising:

-   -   establishing a sample containing an RNA oligonucleotide;    -   contacting said RNA oligonucleotide with a pseudouridine        synthase or a fragment or variant thereof, converting one or        more uridine nucleotide residues in said RNA oligonucleotide        into one or more pseudouridine residues;    -   contacting said pseudouridine residues with a N1-pseudouridine        methyltransferase, or a fragment or variant thereof, forming        N1-methyl-pseudouridine (m1Ψ) residues;    -   digesting said RNA oligonucleotide containing the m1Ψ residues        forming nucleotide mono-phosphates, including m1Ψ-monophosphate        (m1ΨMP);    -   isolating the m1ΨMP from said sample; and    -   optionally regenerating pseudouridine-5′-triphosphate (m1ΨTP)        from said m1ΨMP.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein the step of establishing comprisesisolating said RNA oligonucleotide.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of isolating comprises thestep of isolating a quantity of RNA oligonucleotides from fermentationwaste.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said fermentation waste is selected fromthe group consisting of: bacterial culture waste, yeast culture waste,and food and/or beverage fermentation waste.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said pseudouridine synthase is selectedfrom the group consisting of: SEQ ID NO's. 11-19, or a fragment orvariant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises Nep1 or a fragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said Nep1 comprises a peptide accordingto SEQ ID NO. 8, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises a modified N1-pseudouridine methyltransferase (mNep1).

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said modified mNep1 comprises a modifiedmNep1 wherein 129R and 132R are converted to 129A and 132A.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said modified N1-pseudouridinemethyltransferase comprises a peptide according to SEQ ID NO. 9, or afragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of digesting comprisesdigesting said RNA oligonucleotide with nuclease P1, or5′-Phosphodiesterase (5′-PDase), or a fragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of isolating comprisespurifying m1ΨMP.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of purifying m1ΨMP comprisespurifying m1ΨMP with a weak anion exchange column.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of regenerating comprises thestep of contacting said m1ΨMP with a nucleoside diphosphate kinase (NdK)in the presence of an adenosine-triphosphate (ATP) donor.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said NdK is an adenosyl kinase (AdK)according to: SEQ ID NO. 10, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine further comprising the step of isolating saidm1ΨTP.

In another preferred embodiment, the invention includes a methodproducing pseudouridine further comprising the step of regenerating saidATP donor by contacting adenosine-monophosphate (AMP) is a Polyphosphatekinase (PPK) or enzyme in the presence of inorganic polyphosphate.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said PPK2 is selected from the groupconsisting of: SEQ ID NO's. 4, 6, 21 or a fragment or variant thereof.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein one or more of said steps of theinvention, are performed in an in vitro transcription system.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said step of regenerating is performedin an in vitro transcription system.

In another preferred embodiment, the invention includes a methodproducing pseudouridine wherein said in vitro transcription systemcomprises an in vitro RNA production system.

In one preferred embodiment, the invention includes a system forproducing pseudouridine comprising:

-   -   a sample containing a quantity RNA oligonucleotides;    -   a pseudouridine synthase enzyme or a fragment or variant        thereof, that converts one or more uridine nucleotide residues        in said RNA oligonucleotide unto one or more pseudouridine        residues;    -   a N1-pseudouridine methyltransferase enzyme, or a fragment or        variant thereof, that methylates said pseudouridine residues        forming N1-methyl-pseudouridine (m1Ψ) residues;    -   a nuclease enzyme that digests said RNA oligonucleotide        containing the m1Ψ residues forming nucleotide mono-phosphates,        including m1Ψ-monophosphate (m1ΨMP).    -   a nucleoside diphosphate kinase (NdK) and adenosine-triphosphate        (ATP) donor that regenerates pseudouridine-5′-triphosphate        (m1ΨTP) from said m1ΨMP.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said sample containing a quantity RNAoligonucleotides comprises an isolated sample containing a quantity RNAoligonucleotides.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein the isolated sample comprises anisolated sample from fermentation waste.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said fermentation waste is selected fromthe group consisting of: bacterial culture waste, yeast culture waste,and food and/or beverage fermentation waste.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said pseudouridine synthase is selectedfrom the group consisting of: SEQ ID NO's. 11-19 or a fragment orvariant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises Nep1 or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said Nep1 comprises a peptide accordingto SEQ ID NO. 8, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises a modified N1-pseudouridine methyltransferase (mNep1).

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said modified mNep1 comprises a mNep1wherein 129R and 132R are converted to 129A and 132A.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said modified N1-pseudouridinemethyltransferase comprises a peptide according to SEQ ID NO. 9, or afragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said nuclease is selected from nucleaseP1, or 5′-Phosphodiesterase (5′-PDase), or a fragment or variantthereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising a m1ΨMP purificationapparatus.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said step of m1ΨMP purificationapparatus comprises a weak anion exchange column.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said NdK is an adenosyl kinase (AdK)according to: SEQ ID NO. 10, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine and further comprising an ATP regenerationsystem comprising:

-   -   a polyphosphate kinase (PPK2);    -   adenosyl kinase (AdK);    -   inorganic polyphosphate; and    -   and adenosine monophosphate (AMP).

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said PPK2 is selected from the groupconsisting of: SEQ ID NO's. 4, 6, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said AdK comprises a peptide accordingto SEQ ID NO. 10, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising an in vitro transcriptionsystem.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said in vitro transcription systemcomprises an in vitro RNA production system.

In one preferred embodiment, the invention includes a method forproducing pseudouridine comprising catalyzing the formation ofpseudouridine-5′-monophosphate (ΨMP) from a uracil nucleobase with aribose-5-phosphate with a pseudouridine-5′-phosphate glycosidase (PsuG)enzyme.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said PsuG is selected from: SEQ ID NO.1-2, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein the concentration of uracil nucleobaseand ribose-5-phosphate substrates and reaction temperature cause thereverse catalyzation of the substrates by PsuG forming ΨMP.

In another preferred embodiment, the invention includes a method forproducing pseudouridine further comprising the step of generatingpseudouridine-5′-triphosphate (ΨTP) from ΨMP by contacting said ΨTP withPPK2 in the present of an inorganic polyphosphate.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said inorganic polyphosphate comprisessodium hexamethaphosphate.

In another preferred embodiment, the invention includes a method forproducing pseudouridine further comprising the step of methylating saidΨTP forming N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said step of methylating comprisingcontacting said ΨTP with a N1-pseudouridine methyltransferase, or afragment or variant thereof, forming N1-methyl-pseudouridine (m1Ψ).

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises a N1-pseudouridine methyltransferase according to SEQ ID NO.8.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said step of generating comprises thestep of contacting said ΨMP with a nucleoside diphosphate kinase (NdK)in the presence of an adenosine-triphosphate (ATP) donor.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said NdK is an adenosyl kinase (AdK)according to: SEQ ID NO. 10, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a method forproducing pseudouridine further comprising the step of isolating saidΨTP.

In another preferred embodiment, the invention includes a method forproducing pseudouridine further comprising the step of regenerating saidATP donor by contacting adenosine-monophosphate (AMP) is a Polyphosphatekinase (PPK) or enzyme in the presence of inorganic polyphosphate.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said PPK2 is selected from the groupconsisting of: SEQ ID NO's. 4, 6, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein one or more of said steps of theinvention are performed in an in vitro transcription system.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said step of regenerating is performedin an in vitro transcription system.

In another preferred embodiment, the invention includes a method forproducing pseudouridine wherein said in vitro transcription systemcomprises an in vitro RNA production system.

In one preferred embodiment, the invention includes a system forproducing pseudouridine comprising:

-   -   a quantity of pseudouridine-5′-phosphate glycosidase (PsuG)        enzyme; and    -   a substrate comprising:        -   a quantity of uracil nucleobase;        -   a quantity of ribose-5-phosphate;    -   wherein said PsuG catalyzes the formation of        pseudouridine-5′-monophosphate (ΨMP) from said substrate.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said PsuG is selected from: SEQ ID NO.1-2, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein the concentration of uracil nucleobaseand ribose-5-phosphate substrates and reaction temperature cause thereverses catalyzation of the substrates by PsuG forming ΨMP.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising a quantity of PPK2 andinorganic polyphosphate, wherein said PPK2 and inorganic polyphosphatecatalyzed the formation of pseudouridine-5′-triphosphate (ΨTP) from ΨMP.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said inorganic polyphosphate comprisessodium hexamethaphosphate.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising a quantity ofN1-pseudouridine methyltransferase, or a fragment or variant thereof,that methylates said ΨTP forming N1-methyl-pseudouridine-5′-triphosphate(m1ΨTP).

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said N1-pseudouridine methyltransferasecomprises a N1-pseudouridine methyltransferase according to SEQ ID NO.8.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising an ATP regeneration systemcomprising:

-   -   a polyphosphate kinase (PPK2);    -   adenosyl kinase (AdK);    -   inorganic polyphosphate; and    -   and adenosine monophosphate (AMP).

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said NdK is an adenosyl kinase accordingto: SEQ ID NO. 10, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said PPK2 is selected from the groupconsisting of: SEQ ID NO's. 4, 6, or a fragment or variant thereof.

In another preferred embodiment, the invention includes a system forproducing pseudouridine further comprising an in vitro transcriptionsystem.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said step of regenerating is performedin an in vitro transcription system.

In another preferred embodiment, the invention includes a system forproducing pseudouridine wherein said in vitro transcription systemcomprises an in vitro RNA production system.

In another preferred embodiment, the invention includes an isolatednucleotide sequence encoding a peptide selected from the groupconsisting of: SEQ ID NO's. 1-21, or a combination, fragment, or variantof the same.

In another preferred embodiment, the invention includes an isolatedexpression vector having a nucleotide sequence, operably linked to apromoter, encoding a peptide selected from the group consisting of: toSEQ ID NO's. 1-21, or a combination, fragment or variant of the same.

1. A system for the production of pseudouridine compounds comprising: asubstrate comprising a quantity of isolated uracil nucleobase, and aquantity of isolated ribose-5-phosphate; a quantity of isolatedpseudouridine-5′-phosphate glycosidase (PsuG) enzyme, or a fragment orvariant thereof, wherein said PsuG catalyzes the formation ofpseudouridine-5′-monophosphate (ΨMP) from the uracil nucleobase with aribose-5-phosphate; a protecting agent that reacts with the hydroxylgroups of said ΨMP forming a protected ΨMP; a methylating agent thatreacts with the protected ΨMP, forming a protectedN1-methyl-pseudouridine-5′-monophosphate (protected m1ΨMP) compound; adeprotection agent that reacts with the protected m1ΨMP formingN1-methyl-pseudouridine-5′-monophosphate (m1ΨMP); and a phosphorylatingagent that catalyzes the sequential phosphorylation of m1ΨMP formingN1-methyl-pseudouridine-5′-diphosphate (m1ΨDP), andN1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).
 2. The system of claim1, wherein said protecting agent comprises an N-silyl compound.
 3. Thesystem of claim 2, wherein said N-silyl compound compriseshexamethyldisilazane (HMDS), wherein said HMDS reacts with the ΨMPforming a protected ΨMP.
 4. The system of claim 1, wherein saidmethylating agent comprises iodomethane.
 5. The system of claim 1,wherein said deprotection agent comprises a quantity of ammonia andmethanol.
 6. The system of claim 1, wherein said phosphorylating agentcomprises a quantity of Polyphosphate kinase (PPK2), or a fragment orvariant thereof, and a quantity of inorganic polyphosphate, wherein saidPPK2 catalyzes the sequential phosphorylation of m1ΨMP to form m1ΨDP andm1ΨTP.
 7. The system of claim 6, wherein said concentration of inorganicpolyphosphate is in excess, such that it promotes the forward reactionof the sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP. 8.The system of claim 6, wherein the sequential phosphorylation of m1ΨMPby PPK is performed at a temperature, in the presence of excessinorganic polyphosphate generating reaction conditions that causes theforward reaction of the sequential phosphorylation of m1ΨMP to formm1ΨDP and m1ΨTP.
 9. The system of claim 6, wherein said PPK comprises aPPK from a thermophilic bacteria.
 10. The system of claim 9, whereinsaid thermophilic bacteria is selected from Deinococcus geothermalis,Deinococcus radiodurans, or Meiothermus Tuber.
 11. The system of claim6, wherein said PPK comprises a sequence according to SEQ ID NO. 4, 6,21, or a sequence having at least 80% homology with SEQ ID NO. 4, 6, or21.
 12. The system of claim 1, wherein said PsuG comprises a PsuG from aan enteric bacteria, or a thermophilic bacteria.
 13. The system of claim12, wherein said thermophilic bacteria comprises Deinococcusgeothermalis, and said enteric bacteria comprises Escherichia coli. 14.The system of claim 1, wherein said PsuG comprises a sequence selectedfrom SEQ ID NO. 1, 3, or a sequence having at least 80% homology withSEQ ID NO. 1 or
 3. 15. The system of claim 1, further comprising: aquantity of isolated Ribokinase (RbsK) enzyme, or a fragment or variantthereof, and a quantity of ribose; wherein said RbsK catalyzes theformation of said ribose-5-phosphate from said ribose in the presence ofan adenosine-triphosphate (ATP) donor, which donates a phosphate groupto said ribose to form the ribose-5-phosphate and anadenosine-diphosphate (ADP).
 16. The system of claim 15, furthercomprising a quantity of Polyphosphate kinase (PPK2), or a fragment orvariant thereof, wherein said PPK2 catalyzes the regeneration of ATPfrom the ADP in the presence of inorganic polyphosphate.
 17. The systemof claim 15, wherein said RbsK comprises an RbsK from a thermophilicbacteria.
 18. The system of claim 17, wherein said thermophilic bacteriacomprises Deinococcus geothermalis
 19. The system of claim 15, whereinsaid RbsK comprises a sequence according to SEQ ID NO. 20, or a sequencehaving at least 80% homology with SEQ ID NO.
 20. 20-22. (canceled) 23.The system of claim 1, wherein the concentration of said uracilnucleobase and ribose-5-phosphate substrates and reaction temperaturecause the reverse catalyzation of the substrates by the PsuG forming theΨMP. 24-107. (canceled)
 108. A system for the production ofpseudouridine compounds comprising: a substrate comprising a quantity ofisolated uracil nucleobase, and a quantity of isolatedribose-5-phosphate; a quantity of isolated pseudouridine-5′-phosphateglycosidase (PsuG) enzyme, or a fragment or variant thereof, whereinsaid PsuG catalyzes the formation of pseudouridine-5′-monophosphate(ΨMP) from the uracil nucleobase with a ribose-5-phosphate; and aprotecting agent that reacts with the hydroxyl groups of said ΨMPforming a protected ΨMP.
 109. A system for the production ofpseudouridine compounds comprising: a substrate comprising a quantity ofisolated uracil nucleobase, and a quantity of isolatedribose-5-phosphate; a quantity of isolated pseudouridine-5′-phosphateglycosidase (PsuG) enzyme, or a fragment or variant thereof, whereinsaid PsuG catalyzes the formation of pseudouridine-5′-monophosphate(ΨMP) from the uracil nucleobase with a ribose-5-phosphate; a protectingagent that reacts with the hydroxyl groups of said ΨMP forming aprotected ΨMP; and a methylating agent that reacts with the protectedΨMP, forming a protected N1-methyl-pseudouridine-5′-monophosphate(protected m1ΨMP) compound.
 110. A system for the production ofpseudouridine compounds comprising: a substrate comprising a quantity ofisolated uracil nucleobase, and a quantity of isolatedribose-5-phosphate; a quantity of isolated pseudouridine-5′-phosphateglycosidase (PsuG) enzyme, or a fragment or variant thereof, whereinsaid PsuG catalyzes the formation of pseudouridine-5′-monophosphate(ΨMP) from the uracil nucleobase with a ribose-5-phosphate; a protectingagent that reacts with the hydroxyl groups of said ΨMP forming aprotected ΨMP; a methylating agent that reacts with the protected ΨMP,forming a protected N1-methyl-pseudouridine-5′-monophosphate (protectedm1ΨMP) compound; and a deprotection agent that reacts with the protectedm1ΨMP forming N1-methyl-pseudouridine-5′-monophosphate (m1ΨMP).